CN115386537B

CN115386537B - Chemically defined medium additives for Vero cells and their use in culturing cells and amplifying viruses

Info

Publication number: CN115386537B
Application number: CN202211330594.3A
Authority: CN
Inventors: 陈文庆; 赵洪磊; 徐舸辰; 王杰勇; 浦勇; 类成霞
Original assignee: Tianxinhe Suzhou Biotechnology Co ltd
Current assignee: Tianxinhe Suzhou Biotechnology Co ltd
Priority date: 2022-10-28
Filing date: 2022-10-28
Publication date: 2023-05-02
Anticipated expiration: 2042-10-28
Also published as: CN115386537A

Abstract

The application provides a chemically defined culture medium additive which can be effectively used for culturing Vero cells, does not contain components such as serum, protein and the like, and can be used together with a MEM culture medium to effectively realize cell expansion or culture and effectively shorten multiplication time; in addition, when the Vero cell or virus amplification, especially the amplification production of virus vaccine is carried out, the obtained product does not contain other protein components, can reduce the safety risk of animal-derived additives such as serum and the like, reduces the pressure of other proteins on virus vaccine purification preparations in the production process, and is more suitable for industrial production.

Description

Chemically defined medium additives for Vero cells and their use in culturing cells and amplifying viruses

Technical Field

The present application relates to the field of biotechnology, in particular, the present application relates to a chemically defined medium additive for Vero cells, and its use in methods of culturing cells and amplifying viruses, more particularly, the present application relates to a chemically defined medium additive for culturing Vero cells, a method of amplifying Vero cells or viruses, and a vaccine.

Background

The novel coronavirus pneumonia inactivated vaccine (also called Vero cell) is a novel coronavirus vaccine, and is suitable for preventing diseases caused by novel coronavirus infection. In the culturing of Vero cells under the condition that it is often necessary to add protein additives such as Fetal Calf Serum (FCS) or protein hydrolysates derived from animals, plants or yeasts to the culture medium, other protein components (e.g., fetal calf serum, protein hydrolysates, etc.) are contained in the obtained Vero cells, and the Vero cells are injected into the animal organism, there is a risk of eliciting unnecessary immune responses. In addition, protein hydrolysates derived from animals, plants or yeasts have large differences between batches, which are detrimental to cell proliferation and protein expression in each cell. Therefore, development of a novel chemolimiting medium additive for culturing Vero cells continues.

Disclosure of Invention

The present application aims to solve, at least to some extent, one of the above technical problems or at least to provide a useful commercial choice. To this end, an object of the present application is to propose a chemically defined medium additive that can be effectively used for culturing Vero cells.

Thus, in one aspect of the present application, there is provided a chemically defined medium additive for culturing Vero cells, said additive being complexed with MEM medium, said additive comprising:

41 parts by weight of L-alanine,

98 parts by weight of L-arginine hydrochloride,

32.5 parts by weight of L-aspartic acid,

31 parts by weight of L-asparagine,

8 parts by weight of L-cysteine hydrochloride monohydrate,

27.5 parts by weight of L-cystine dihydrochloride,

47.5 parts by weight of L-glutamic acid,

292.5 parts by weight of L-glutamine,

23.08 parts by weight of glycine,

24.5 parts by weight of L-histidine hydrochloride monohydrate,

14 parts by weight of L-hydroxyproline,

36 parts by weight of L-isoleucine,

39 parts by weight of L-leucine,

50 parts by weight of L-lysine hydrochloride,

15.5 parts by weight of L-methionine,

21 parts by weight of L-phenylalanine,

25.5 parts by weight of L-proline,

42 parts by weight of L-serine,

30 parts by weight of L-threonine,

7.5 parts by weight of L-tryptophan,

32 parts by weight of L-valine,

0.585 The weight portions of the reduced glutathione are as follows,

0.698 Ferrous sulfate heptahydrate in parts by weight,

0.765 Zinc sulfate heptahydrate in parts by weight,

3.375 The weight portions of the vitamin C are as follows,

39.713 The choline chloride is prepared from (by weight parts),

3.668 The weight portions of the D-calcium pantothenate,

3.544 The folic acid is added in the weight portions,

42.307 The inositol comprises the following components in parts by weight,

0.056 Ferric nitrate nonahydrate in the weight portions,

1.406 The lithium chloride is added in the weight portions,

6.188 The citric acid is added in the weight portions,

0.563 Pyridoxal hydrochloride in a weight portion,

5.378 The weight portions of the nicotinamide are calculated as follows,

2.909 The weight portions of the vitamin B6,

3.173 The weight portions of the vitamin B1,

7.178 The weight portions of the vitamin B12 are as follows,

0.203 The weight portions of the D-biotin,

4.028 The weight portions of the hypoxanthine are as follows,

0.140 The weight portions of the linoleic acid are as follows,

0.473 The weight portions of the lipoic acid are calculated,

0.135 1, 4-butanediamine dihydrochloride in parts by weight,

0.518 The weight portions of the vitamin B2,

0.878 The weight portions of the thymidine are that,

0.585 The weight portions of the para-aminobenzoic acid,

2.250 The weight portions of the recombinant human insulin,

0.008 parts by weight of hydrocortisone,

1000. f68 in parts by weight,

2000. the HEPES is added in parts by weight,

3000. the weight portions of the anhydrous dextrose,

0.000150056 parts by weight of aluminum chloride,

0.000431411 parts by weight of barium acetate,

0.00018757 parts by weight of cobalt chloride hexahydrate,

1.50056E-05 parts by weight of manganese chloride tetrahydrate,

0.00056271 parts by weight of sodium fluoride,

0.000131299 parts by weight of stannous chloride,

0.000131299 parts by weight of cadmium chloride,

5.6271E-05 parts by weight of germanium dioxide,

0.00375 parts by weight of sodium selenite,

0.00075028 sodium metasilicate nonahydrate,

1.8757E-05 parts by weight of nickel chloride,

3.7514E-05 parts by weight of ammonium metavanadate,

4.69E-06 parts by weight of potassium bromide,

1.50056E-05 parts by weight of potassium iodide,

7.50E-07 parts by weight of rubidium chloride,

9.3785E-06 parts by weight of ammonium molybdate,

5.63E-07 parts by weight of silver nitrate.

The inventor optimizes and adjusts the chemically defined culture medium additive (simply referred to as additive) for culturing Vero cells through a large number of experiments, and does not find the rule among all the formulas of the chemically defined culture medium additive, and the result is unpredictable. However, when the additive is adopted to culture Vero cells by a chemically defined culture medium obtained by matching the additive with a MEM culture medium, cell expansion or cell culture can be effectively realized, and the multiplication time is shortened; in addition, the chemically defined culture medium additive does not contain serum or protein, and is used for amplifying Vero cells or viruses, especially virus vaccines, and the obtained product does not contain other protein components, so that the safety risk of animal-derived additives such as serum and the like can be reduced, the pressure of other proteins on virus vaccine purification preparations in the production process is reduced, and the method is more suitable for industrial production.

The "chemically defined medium" herein refers to a medium in which the chemical components are completely defined, and the composition and concentration are fixed. The chemically defined medium containing the additive has the advantages of small batch-to-batch difference, strong repeatability, stable result, no animal source risk and the like.

According to an embodiment of the present application, the MEM medium is formulated as follows:

raw material name	mg/L
		L-alanine	8.9
L-arginine hydrochloride	126
		L-asparagine	13.2
L-aspartic acid	13.2
		L-cystine dihydrochloride	31
L-glutamic acid	14.7
		L-glutamine	292
Glycine (Gly)	7.5
		L-histidine hydrochloride monohydrate	42
L-isoleucine	52
		L-leucine	52
L-lysine hydrochloride	73
		L-methionine	15
L-phenylalanine	32
		L-proline	11.5
L-serine	10.5
		L-threonine	48
L-tryptophan	10
		Anhydrous L-tyrosine disodium salt	52
L-valine	46
		Phenol red	10
Calcium chloride dihydrate	265
		Potassium chloride	400
Anhydrous magnesium sulfate	98
		Sodium chloride	6800
Sodium dihydrogen phosphate monohydrate	140
		D-Anhydrous glucose	1000
D-pantothenic acid calcium salt	1
		Folic acid	1
Choline chloride	1
		Inositol (inositol)	2
Nicotinamide	1
		Pyridoxal hydrochloride	1
Vitamin B2	0.1
		Vitamin B1	1

In another aspect of the present application, the present application provides a method of expanding Vero cells comprising: inoculating Vero cells in MEM medium, said MEM medium being supplemented with the aforementioned additives; culturing the Vero cells so as to achieve expansion of the Vero cells. According to the embodiment of the application, the method has higher amplification efficiency of the Vero cells.

According to the embodiment of the application, the final concentration of L-alanine is 41mg/L, the final concentration of L-arginine hydrochloride is 98mg/L, the final concentration of L-aspartic acid is 32.5mg/L, the final concentration of L-asparagine is 31mg/L, the final concentration of L-cysteine hydrochloride-water is 8mg/L, the final concentration of L-cystine dihydrochloride is 27.5mg/L, the final concentration of L-glutamic acid is 47.5mg/L, the final concentration of L-glutamine is 292.5mg/L, the final concentration of glycine is 23.08mg/L, the final concentration of L-histidine hydrochloride-water is 24.5mg/L, the final concentration of L-hydroxyproline is 14mg/L, the final concentration of L-isoleucine was 36mg/L, the final concentration of L-leucine was 39mg/L, the final concentration of L-lysine hydrochloride was 50mg/L, the final concentration of L-methionine was 15.5mg/L, the final concentration of L-phenylalanine was 21mg/L, the final concentration of L-proline was 25.5mg/L, the final concentration of L-serine was 42mg/L, the final concentration of L-threonine was 30mg/L, the final concentration of L-tryptophan was 7.5mg/L, the final concentration of L-valine was 32mg/L, the final concentration of reduced glutathione was 0.585 mg/L, the final concentration of ferrous sulfate heptahydrate was 0.698mg/L, the final concentration of zinc sulfate heptahydrate was 0.765 mg/L, the final concentration of vitamin C is 3.375 mg/L, the final concentration of choline chloride is 39.713 mg/L, the final concentration of D-calcium pantothenate is 3.668 mg/L, the final concentration of folic acid is 3.544 mg/L, the final concentration of inositol is 42.307mg/L, the final concentration of ferric nitrate nonahydrate is 0.056 mg/L, the final concentration of lithium chloride is 1.406 mg/L, the final concentration of citric acid is 6.188 mg/L, the final concentration of pyridoxal hydrochloride is 0.563mg/L, the final concentration of nicotinamide is 5.378 mg/L, the final concentration of vitamin B6 is 2.909mg/L, the final concentration of vitamin B1 is 3.173 mg/L, the final concentration of vitamin B12 is 7.178 mg/L, the final concentration of D-biotin is 0.203 7.178 mg/L, the final concentration of hypoxanthine is 4.028/L, the final concentration of linoleic acid is 7.178 mg/L, and the final concentration of linoleic acid is 0.140/L, the final concentration of lipoic acid is 0.473 7.178 mg/L, the final concentration of 1, 4-butanediamine dihydrochloride is 0.135 7.178 mg/L, the final concentration of vitamin B2 is 0.518 7.178 mg/L, the final concentration of thymidine is 0.878 7.178 mg/L, the final concentration of p-aminobenzoic acid is 0.585 7.178 mg/L, the final concentration of recombinant human insulin is 2.250 7.178 mg/L, the final concentration of hydrocortisone is 0.008mg/L, the final concentration of F68 is 1000 7.178 mg/L, the final concentration of HEPES is 2000 7.178 mg/L, the final concentration of anhydrous glucose is 3000 7.178 mg/L, the final concentration of aluminum chloride is 7.178 mg mg/L, the final concentration of barium acetate is 7.178 mg mg/L, the final concentration of cobalt chloride hexahydrate is 7.178 mg mg/L, the final concentration of manganese chloride tetrahydrate is 7.178 mg E-05mg/L, the final concentration of sodium fluoride is 0.00056271mg/L, the final concentration of stannous chloride is 0.000131299mg/L, the final concentration of cadmium chloride is 0.000131299mg/L, the final concentration of germanium dioxide is 5.6271E-05mg/L, the final concentration of sodium selenite is 0.00375mg/L, the final concentration of sodium metasilicate nonahydrate is 0.00075028mg/L, the final concentration of nickel chloride is 1.8757E-05mg/L, the final concentration of ammonium metavanadate is 3.7514E-05mg/L, the final concentration of potassium bromide is 4.69E-06mg/L, the final concentration of potassium iodide is 1.50056E-05mg/L, the final concentration of rubidium chloride is 7.50E-07mg/L, the final concentration of ammonium molybdate is 9.3785E-06mg/L, and the final concentration of silver nitrate is 5.63E-07mg/L.

According to an embodiment of the present application, the culture is an adherent culture.

In yet another aspect of the present application, the present application provides a method of amplifying a virus comprising: the virus was amplified according to the method for amplifying Vero cells described previously.

According to an embodiment of the present application, the virus comprises at least one selected from rabies, influenza, coronavirus, bursa of fabricius, dengue, encephalitis, poxvirus, orthomyxovirus, paramyxovirus, retrovirus, togavirus, flaviviridae, enterovirus, picornavirus, monoshaid virus, herpesvirus, adenovirus, vaccinia virus, SARS virus, influenza a virus, influenza b virus, lentivirus, ross river virus, west nile virus, yellow fever virus, FSME virus, and hepatitis a virus.

In a further aspect of the present application, the present application proposes a vaccine obtained by the following method: amplifying the virus according to the method; and subjecting the virus to an attenuation or inactivation treatment the vaccine is prepared according to the method described previously. According to the embodiment of the application, the vaccine does not contain other protein components, so that the safety risk of animal-derived additives such as serum and the like can be reduced, and the vaccine has the advantage of high safety.

In addition, in a further aspect of the application, the application also provides the use of the vaccine in the preparation of medicaments for treating or preventing virus-related diseases.

Additional aspects and advantages of the application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the application.

Detailed Description

The following detailed description of embodiments of the present application is exemplary and intended to be used to explain the present application and should not be taken as limiting the present application.

In this document, the terms "comprise" or "include" are used in an open-ended fashion, i.e., to include what is indicated in the present application, but not to exclude other aspects.

In this context, the term "treatment" refers to the use to obtain a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing the disease or symptoms thereof, and/or may be therapeutic in terms of partially or completely curing the disease and/or adverse effects caused by the disease. As used herein, "treating" encompasses diseases in mammals, particularly humans, including: (a) Preventing the occurrence of a disease or disorder in an individual susceptible to the disease but not yet diagnosed with the disease; (b) inhibiting disease, e.g., arresting disease progression; or (c) alleviating a disease, e.g., alleviating symptoms associated with a disease. As used herein, "treating" or "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, alleviate, ameliorate, reduce or inhibit a disease in the individual, including, but not limited to, administration of a drug comprising a compound described herein to an individual in need thereof.

The following will explain the scheme of the present application in conjunction with examples. Those skilled in the art will appreciate that the following examples are illustrative of the present application and should not be construed as limiting the scope of the present application. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Example 1

1. Preparing culture medium

Commercially available MEM media were obtained, see in particular table 1. Additives were added to MEM media according to table 2 to give medium 1 and medium 2, respectively, wherein medium 1 was a serum-free medium prepared with the additives of the present application and medium 2 was a serum-free medium prepared with the control additives. A medium of mem+10% serum was prepared as a serum medium, while a commercially available medium (healthy Vero CD medium) was prepared as a control.

Table 1: MEM culture medium formulation

Table 2: formulation and addition amount of each additive in culture medium 1 and culture medium 2

Table 2 follow:

2. cell culture and detection

After medium 1, medium 2, serum medium and commercial medium were previously obtained, vero cells were cultured in the following manner:

vero cells (P157) were grown according to 5X 10 ⁶ Inoculating cells into T75 square bottle (ventilation cap), placing at 37deg.C, 5% CO ₂ Is cultured in an incubator of (a). After 3d culture, digestion and counting with TrypLE ™ Express Enzyme (1X) pancreatin, inoculating into T75 square bottle according to the above cell number, and continuously passaging to calculate cell doubling time (double time, also called DT), which is an important index for judging whether cell growth is vigorous, can be expressed by formula (DT=tXl2/(lgNt-lgN 0)]) And (5) calculating. t is the culture time, N0 is the number of inoculated cells, nt is the number of cells after t time.

3. Results and analysis

The results of cell doubling time measurements for Medium 1, medium 2, serum Medium and commercial Medium are shown in Table 3. As a result, it was found that the average DT time of medium 1 was 30.41 hours, the average DT time of the serum medium was 35.78 hours, and the doubling time was shortened by 17.66% on average, i.e., by 5.37 hours. The components of the additives in the culture medium 1 and the culture medium 2 are the same, but the addition amounts are different, the DT time of the culture medium 2 is increased by 5% compared with the commercial culture medium, and the DT time of the culture medium 1 is shortened by 4.6% compared with the commercial culture medium.

Table 3: results of detection of cell doubling time of Medium 1, medium 2, serum Medium and commercial Medium

In summary, the examples of the present application further demonstrate that the culture medium 1 of the present application is obtained through a large number of experimental screening, and that irregularities can be followed, and that each component of the additive in the culture medium 1 is important for the culture effect (e.g., DT time), especially for significantly shortening the doubling time and improving the culture efficiency of Vero cells.

In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present application. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

Although embodiments of the present application have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the application, and that variations, modifications, alternatives, and variations may be made to the embodiments by those of ordinary skill in the art without departing from the principles and spirit of the application.

Claims

1. A chemically defined medium additive for culturing Vero cells, said additive being complexed with MEM medium, characterized in that said additive consists of: l-alanine at a final concentration of 41mg/L, L-arginine hydrochloride at a final concentration of 98mg/L, L-aspartic acid at a final concentration of 32.5mg/L, L-asparagine at a final concentration of 31mg/L, L-cysteine hydrochloride monohydrate at a final concentration of 8mg/L, L-cystine dihydrochloride at a final concentration of 27.5mg/L, L-glutamic acid at a final concentration of 47.5mg/L, L-glutamine at a final concentration of 292.5mg/L, glycine at a final concentration of 23.08mg/L, L-hydrochloric acid set at a final concentration of 24.5mg/LAmino acid water, L-hydroxyproline with a final concentration of 14mg/L, L-isoleucine with a final concentration of 36mg/L, L-leucine with a final concentration of 39mg/L, L-lysine hydrochloride with a final concentration of 50mg/L, L-methionine with a final concentration of 15.5mg/L, L-phenylalanine with a final concentration of 21mg/L, L-proline with a final concentration of 25.5mg/L, L-serine with a final concentration of 42mg/L, L-threonine with a final concentration of 30mg/L, L-tryptophan with a final concentration of 7.5mg/L, L-valine with a final concentration of 32mg/L, reduced glutathione with a final concentration of 0.585 mg/L, ferrous sulfate heptahydrate with a final concentration of 0.698mg/L, zinc sulfate heptahydrate with a final concentration of 0.765 mg/L, vitamin C with a final concentration of 3. mg/L, choline chloride at a final concentration of 39.713 mg/L, calcium D-pantothenate at a final concentration of 3.668 mg/L, folic acid at a final concentration of 3.544 mg/L, inositol at a final concentration of 42.307mg/L, ferric nitrate nonahydrate at a final concentration of 0.056 mg/L, lithium chloride at a final concentration of 1.406 mg/L, citric acid at a final concentration of 6.188 mg/L, pyridoxal hydrochloride at a final concentration of 0.563mg/L, nicotinamide at a final concentration of 5.378 mg/L, vitamin B6 at a final concentration of 2.909mg/L, vitamin B1 at a final concentration of 3.173 mg/L, vitamin B12 at a final concentration of 7.178 mg/L, D-biotin at a final concentration of 0.203 mg/L, hypoxanthine at a final concentration of 4.028 mg/L, linoleic acid at a final concentration of 0.140 mg/L, lipoic acid at a final concentration of 0.473mg/L, 1, 4-butanediamine dihydrochloride at a final concentration of 0.135 mg/L, vitamin B2 at a final concentration of 0.518 mg/L, thymidine at a final concentration of 0.878 mg/L, p-aminobenzoic acid at a final concentration of 0.585 mg/L, recombinant human insulin at a final concentration of 2.250mg/L, hydrocortisone at a final concentration of 0.008mg/L, F68 at a final concentration of 1000 mg/L, HEPES at a final concentration of 2000 mg/L, anhydrous glucose at a final concentration of 3000 mg/L, aluminum chloride at a final concentration of 0.000150056mg/L, barium acetate at a final concentration of 0.000431411mg/L, cobalt chloride hexahydrate at a final concentration of 0.00018757mg/L, and cobalt chloride at a final concentration of 1.50056 ×10 ^-5 manganese chloride tetrahydrate of mg/L, sodium fluoride of 0.00056271mg/L, stannous chloride of 0.000131299mg/L, cadmium chloride of 0.000131299mg/L and cadmium chloride of 5.6271 ×10 ^-5 mg/L germanium dioxide, sodium selenite with a final concentration of 0.00375mg/L, sodium metasilicate nonahydrate with a final concentration of 0.00075028mg/L, and the likeThe concentration is 1.8757 multiplied by 10 ^-5 mg/L nickel chloride, final concentration 3.7514 ×10 ^-5 mg/L ammonium metavanadate with a final concentration of 4.69×10 ^-6 mg/L potassium bromide, final concentration of 1.50056X 10 ^-5 mg/L potassium iodide at a final concentration of 7.50X10 ^-7 mg/L rubidium chloride, final concentration 9.3785 ×10 ^-6 mg/L ammonium molybdate.

2. A method of expanding Vero cells comprising:

inoculating Vero cells in MEM medium supplemented with the additive of claim 1;

culturing the Vero cells so as to achieve expansion of the Vero cells.

3. The method of claim 2, wherein the culturing is an adherent culturing.

4. A method of amplifying a virus, comprising:

a Vero cell amplified according to the method of claim 2 or 3 for amplifying a virus.

5. The method of claim 4, wherein the virus comprises at least one selected from the group consisting of rabies, influenza, coronavirus, bursa of fabricius, dengue, enterovirus, encephalitis, poxvirus, orthomyxovirus, paramyxovirus, retrovirus, togavirus, flavivirus, picornavirus, papovavirus, herpesvirus, adenovirus, vaccinia virus, lentivirus, ross river virus, west nile virus, yellow fever virus, FSME virus, and hepatitis a virus.

6. The method of claim 4, wherein the virus comprises at least one selected from the group consisting of SARS virus, influenza a virus, influenza b virus.